Severe retinopathy of prematurity: longitudinal observation of disease and screening implications.

PURPOSE: To test the effectiveness of our Canadian retinopathy of prematurity (ROP) screening guidelines as applied to high-risk premature infants. STUDY DESIGN: Retrospective longitudinal cohort study. SUBJECTS: A total of 969 infants were examined longitudinally between 1991 and 2000 and 46 of these infants screened were treated for severe ROP. METHODS: Data from weekly ROP screening examination results were collected from a geographical area and analysed. RESULTS: The average incidence of severe ROP requiring treatment in the population of premature infants eligible for screening was 48.3 per 1000. In all, 46 infants were treated in this cohort. The mean gestational age (GA) was 25.5 weeks of age and the mean birth weight was 750 g. The mean chronological age (CA) and postmenstrual age (PMA) at the time of first screening was 36 days and 30.7 weeks, respectively. The first identification of any ROP in this group was at a mean CA 60 days and PMA of 34.1 weeks. The mean CA and PMA of the first observation of stage 3 were 74 days and 36.3 weeks. The mean CA and PMA at the time of treatment were 86 days and 37.7 week. CONCLUSIONS: Our observations and analysis indicate the following ROP screening recommendations: infants of 28 weeks of GA or less, infants with a GA between 28 weeks and 30 weeks should have a single 'spot examination' at approximately 37 weeks of PMA (or prior to discharge from hospital) to include possible outliers; infants born with a birth weight of 1250 g or less; initial screening examination should be at 31 weeks of PMA or 4 weeks of CA, whichever is later; in the presence of any active ROP, the infant should be followed every 1-2 weeks; and stage 3 should be followed at least every 7 days.

Comparative analysis of dioxin response elements in human, mouse and rat genomic sequences.

Comparative approaches were used to identify human, mouse and rat dioxin response elements (DREs) in genomic sequences unambiguously assigned to a nucleotide RefSeq accession number. A total of 13 bona fide DREs, all including the substitution intolerant core sequence (GCGTG) and adjacent variable sequences, were used to establish a position weight matrix and a matrix similarity (MS) score threshold to rank identified DREs. DREs with MS scores above the threshold were disproportionately distributed in close proximity to the transcription start site in all three species. Gene expression assays in hepatic mouse tissue confirmed the responsiveness of 192 genes possessing a putative DRE. Previously identified functional DREs in well-characterized AhR-regulated genes including Cyp1a1 and Cyp1b1 were corroborated. Putative DREs were identified in 48 out of 2437 human-mouse-rat orthologous genes between -1500 and the transcriptional start site, of which 19 of these genes possessed positionally conserved DREs as determined by multiple sequence alignment. Seven of these nineteen genes exhibited 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated regulation, although there were significant discrepancies between in vivo and in vitro results. Interestingly, of the mouse-rat orthologous genes with a DRE between -1500 and +1500, only 37% had an equivalent human ortholog. These results suggest that AhR-mediated gene expression may not be well conserved across species, which could have significant implications in human risk assessment.

Effect of human dietary exposure levels of genistein during gestation and lactation on long-term reproductive development and sperm quality in mice.

The objective of the present study was to determine the long-term reproductive effects of gestational and lactational exposure (0, 0.1, 0.5, 2.5 and 10 mg/kg/day) to genistein on male mice at levels comparable to or greater than human dietary exposures. Testicular growth, sperm count and motility, and sperm fertilizing ability in vitro was assessed in male offspring on postnatal days (PND) 105 and 315. Selected genes were also examined by real-time PCR to determine whether genistein caused changes in gene expression similar to those previously observed with diethylstilbestrol (DES). No significant treatment-related effects on male offspring body weight, anogenital distance, seminal vesicle weight or testis weight were observed. There were also no significant effects on sperm count, the percent of motile sperm or the number of motile sperm at any age. The in vitro fertilizing ability of epididymal sperm was increased significantly in the high-dose group approximately 17% (P < 0.001) on PND 105 and 315. The results indicate that developmental exposure of mice to genistein at human exposure levels does not induce adverse effects on sperm quality or changes in testicular gene expression similar to DES.

GP3: GenePix post-processing program for automated analysis of raw microarray data.

UNLABELLED: Here we describe an automated and customizable program to correct, filter and normalize raw microarray data captured using GenePix, a commonly used microarray image analysis application. Files can be processed individually or in batch mode for increased throughput. User defined inputs specify the stringency of data filtering and the method and conditions of normalization. The output includes gene summaries for replicate spots and descriptive statistics for each experiment. The source code (Perl) can also be adapted to handle raw data output from other image analysis applications. AVAILABILITY: http://bch.msu.edu/~zacharet/microarray/GP3.html

Effects of gestational and lactational exposure to Aroclor 1242 on sperm quality and in vitro fertility in early adult and middle-aged mice.

The objective of this study was to examine the effects of gestational and lactational exposure to Aroclor 1242 (0, 10, 25, 50, and 100 mg/kg-bw) on male fertility. Doses were administered to C57BL6 female mice orally every two days from two weeks before mating, during mating, and through gestation until postnatal day 21. Male B6D2F1 offspring were examined for anogenital distance, organ development, epididymal sperm count, sperm motility, and in vitro fertility at 16 and 45 weeks of age. Stomach samples of pups nursing from PCB-treated mothers in the 50 mg/kg dose group were analyzed for PCBs and chlorobiphenylols by high resolution gas chromatography coupled with low resolution mass spectrometry. It was estimated that the nursing pups were exposed to 0.2, 0.6, 1.2, and 2.4 mg/kg/day total PCBs in the 10, 25, 50, and 100 mg/kg dose groups, respectively. This exposure level approaches the maximum FDA recommended levels for PCBs in food and breast milk. The composition of the PCBs in the stomach samples was different from the parent mixture, as there was a higher proportion of heavily chlorinated congeners, as well as chlorobiphenylols. Anogenital distance at weaning, and liver, thymus, and testes weight at 16 and 45 weeks of age were not affected by PCB exposure. Epididymal sperm velocity and linearity were significantly increased in the 25 mg/kg dose group at 16 weeks of age. Sperm count was increased by 36% in this dose group (P = 0.06). By 45 weeks of age, average sperm count in this dose group was similar to that of controls. With the exception of the 50 mg/kg dose group at 16 weeks of age, sperm fertilizing ability in vitro was significantly decreased in all PCB-exposed groups at 16 and 45 weeks of age. These results suggest that fertility in the adult mouse is susceptible to developmental exposure to Aroclor 1242 and is independent of testis weight or epididymal sperm count.

Sugar-based tertiary amino gemini surfactants with a vesicle-to-micelle transition in the endosomal pH range mediate efficient transfection in vitro.

Novel reduced sugar gemini amphiphiles linked through their tertiary amino head groups via alkyl spacers of 4 or 6 carbons, and with varying (unsaturated) alkyl tail lengths of 12--18, have been synthesized and tested for transfection in vitro in an adherent Chinese hamster ovary cell line (CHO-K1). Transfection efficiencies peaked at 2.7 times that of the commercial standard Lipofectamine Plus/2000 for pure solutions of the compound bearing unsaturated (oleyl) alkyl tails. For those compounds bearing saturated alkyl tails, transfection efficiency peaked at a tail length of 16, at a level similar to Lipofectamine Plus/2000. All of the amphiphiles formed bilayer vesicles at physiological pH. Some of the amino groups at the surface were protonated, and vesicles therefore bore a positive charge. Increased protonation with reduced pH resulted in greatly increased monomer solubility and a morphology change from vesicle to micelle at characteristic pH values, dependent on the tail length. For the compounds promoting high transfection efficiency, this characteristic pH was within the range found in the endosomal compartment (7.4--4.0). Formation of mixed micelles between gemini surfactant and membrane phospholipids at reduced pH may therefore provide a method of endosome rupture and subsequent escape of entrapped DNA, thus discarding the need for extra fusogenic or endosomolytic agents. The positive charge on the vesicles at physiological pH drives the colloidal association with DNA. Small angle X-ray scattering measurements indicate that lamellar aggregates are formed, which have a d spacing of 48--54 A. Preliminary differential scanning calorimetric measurements suggest that reduction of pH causes a disordering of the hydrocarbon region of the DNA-surfactant complex.

Challenges and limitations of gene expression profiling in mechanistic and predictive toxicology.

RNA and protein expression profiling technologies have revolutionized how toxicologists can study the molecular basis of adverse effects of chemicals and drugs. It is expected that these new technologies will afford efficient and high-throughput means to delineate mechanisms of action and predict toxicity of unknown agents. To reach these goals, a more thorough understanding of the constraints of the methodology is needed to design genome-scale studies and to interpret the vast amount of data collected. This paper addresses some of the limitations and uncertainties of gene expression profiling in mechanistic and predictive toxicology with respect to the expectations of toxicogenomics. The challenges associated with interpreting information from large-scale gene expression experiments in vivo is also discussed.

Assessment of clone identity and sequence fidelity for 1189 IMAGE cDNA clones.

This report documents the error rate in a commercially distributed subset of the IMAGE Consortium mouse cDNA clone collection. After isolation of plasmid DNA from 1189 bacterial stock cultures, only 62. 2% were uncontaminated and contained cDNA inserts that had significant sequence identity to published data for the ordered clones. An agarose gel electrophoresis pre-screening strategy identified 361 stock cultures that appeared to contain two or more plasmid species. Isolation of individual colonies from these stocks demonstrated that 7.1% of the original 1189 stocks contained both a correct and an incorrect plasmid. 5.9% of the original 1189 stocks contained multiple, distinct, incorrect plasmids, indicating the likelihood of multiple contaminating events. While only 739 of the stocks purchased contained the desired cDNA clone, agarose gel pre-screening, colony isolation and similarity searching of dbEST allowed for the identification of an additional 420 clones that would have otherwise been discarded. Considering the high error rate in this subset of the IMAGE cDNA clone set, the use of sequence verified clones for cDNA microarray construction is warranted. When this is not possible, pre-screening non-sequence verified clones with agarose gel electrophoresis provides an inexpensive and efficient method to eliminate contaminated clones from the probe set.

An ELISA for brown trout (Salmo trutta) vitellogenin and its use in bioassays for environmental estrogens.

An enzyme linked immunosorbent assay (ELISA) was developed for the detection of the egg yolk precursor vitellogenin (Vg) in plasma of brown trout (Salmo trutta). Purified Vg from a 17 beta-estradiol-induced trout was used as the competing antigen in the ELISA which is based on polyclonal antibodies. The ELISA's performance was optimized and characterized. The assay's working range was (25-500 ng ml-1), its sensitivity was (10.5 ng ml-1), and it had an intra-assay coefficient of variation of less than 10% between 30 and 1000 ng ml-1. The ELISA was used in bioassays for the detection of environmental estrogens, including estrogen mimics, in whole and fractionated industrial waste waters. Those bioassays were based on intraperitoneal (i.p.) injection-, static renewal-, and flow through exposure systems. The response threshold of both bioassays is limited to 1-2 micrograms ml-1 Vg by a low level plasma interference that was regularly detected in plasma from non-induced male fish. The responsiveness of the bioassays was characterized using progressive doses of 17 beta-estradiol. The i.p.-based assay, which was responsive to at least 100 micrograms kg-1 of 17 beta-estradiol, was used to screen extracts of pulp mill effluent and black liquor for estrogenic effects. Neither extract induced Vg in our assay. The i.p. assay was also used to test 4-tert-octylphenol (OP) and the PAH derivative, retene, for estrogenic activity. OP induced Vg in the i.p.-exposed fish; no Vg induction was detected in the retene-exposed fish. The static renewal bioassay, which was responsive to at least 0.1 microgram ml-1 of 17 beta-estradiol over a 15-day exposure period, was used to screen whole pulp mill effluents for estrogenic effects. No Vg induction was detected in the effluent-treated fish.

Examination of the in vitro and in vivo estrogenic activities of eight commercial phthalate esters.

The estrogenic activities of eight phthalate esters (i.e., di-2-ethylhexyl, di-n-butyl (DBP), butylbenzyl (BBP), di-hexyl (DHP), diiso-heptyl, di-n-octyl, diiso-nonyl, diiso-decyl) were investigated in vitro using estrogen receptor (ER) competitive ligand-binding and mammalian- and yeast-based gene expression assays. In vivo, their effects on uterine wet weight and vaginal cell cornification using ovariectomized Sprague-Dawley rats were assessed. DBP, BBP, and DHP weakly competed with 17 beta-estradiol (E2) for binding to the ER in competitive ligand-binding assays. In gene expression assays using MCF-7 cells transiently transfected with the Gal4-human estrogen receptor construct, Gal4-HEGO, and the Gal4-regulated luciferase reporter gene, 17m5-G-Luc, 10 microM DBP, BBP, or DHP exhibited 36, 42, and 20% activity, respectively, when compared to the 100% response observed with 10 nM E2. Only BBP was found to induce luciferase activity (32%) in HeLa cells stably transfected with Gal4-HEGO and 17m5-G-Luc constructs and to impart minimal ER-mediated viability to the E2-dependent recombinant yeast strain, PL3, on selective medium. No significant responses were observed with the other phthalate esters in any of the in vitro assays. In vivo, none of the eight phthalate esters reproducibly induced significant increases in uterine wet weight in immature ovariectomized Sprague-Dawley rats treated with oral doses of 20, 200, or 2000 mg/kg of phthalate ester. In addition, treatment with phthalate esters at the same doses did not affect the degree of vaginal epithelial cell cornification in mature ovariectomized rats. These results indicate that only selected phthalate esters (i.e., DBP, BBP, and DHP) exhibit weak ER-mediated activity in some in vitro assays at high concentrations but none of the eight phthalate esters elicited in vivo estrogenic responses based upon results obtained from uterotrophic and vaginal cornification assays.

Examination of the estrogenicity of 2,4,6,2',6'-pentachlorobiphenyl (PCB 104), its hydroxylated metabolite 2,4,6,2',6'-pentachloro-4-biphenylol (HO-PCB 104), and a further chlorinated derivative, 2,4,6,2',4',6'-hexachlorobiphenyl (PCB 155).

Several studies have reported that polychlorinated biphenyls (PCBs) exhibit estrogenic activity; however, it is not clear if these responses are associated with the polychlorinated hydrocarbon or its hydroxylated metabolite. In order to further test this hypothesis, a battery of in vitro and in vivo assays were used to investigate the estrogenic and antiestrogenic activities of 2,4,6,2',6'-pentachlorobiphenyl (PCB 104), its para-hydroxylated derivative 2,4,6,2',6'-pentachloro-4-biphenylol (HO-PCB 104), and its para-chlorinated derivative 2,4,6,2',4',6'-hexachlorobiphenyl (PCB 155). PCB 104 was found to 1) compete with tritiated 17beta-estradiol (E2) for binding to the mouse uterine estrogen receptor (ER); 2) induce gene expression in MCF-7 human breast cancer cells transiently transfected with the Gal4-human ER chimeric construct (Gal4-HEGO) and the Gal4-regulated luciferase reporter gene (17m5-G-Luc); and 3) increase MCF-7 cell proliferation in a dose-dependent manner. HO-PCB 104 exhibited greater estrogenic activity than PCB 104 in the in vitro assays examined. However, gas chromatographic-mass spectrophotometric analysis of extracts prepared from MCF-7 cells incubated with PCB 104 failed to detect the presence of the expected major metabolite HO-PCB 104. The estrogenic activity of the para-chlorinated derivative, PCB 155, was minimal compared to PCB 104 and HO-PCB 104, but it did exhibit significant antiestrogenic activity following co-treatment with 1 nM E2. Co-treatment of PCB 104 with 1 nM E2 had no effect on reporter gene expression compared to E2 alone, while 10 microM HO-PCB 104 exhibited additivity with 1 nM E2. At a dose of 202 mg/kg,PCB 104 increased uterine wet weight in ovariectomized CD-1 mice and induced vaginal epithelial cell cornification at 202, 16, and 1.7 mg/kg in a dose-dependent manner. These studies demonstrate that in addition to the hydroxylated metabolites, selected parent PCB congeners may also exhibit estrogenic and antiestrogenic activities.

Sequential feeding of whole wheat to growing broiler chickens.

1. Four broiler feeding trials were performed to examine the suitability of a whole wheat sequential feeding regimen for commercial broiler production. The sequential feeding programme gave a continuous cycle of ad libitum access to only whole wheat followed by the same time of access to only a pelleted diet. The pelleted diet provided a concentration of nutrients to balance that provided by the whole wheat. This was called a balancer diet. 2. The first trial used 144 cage-reared broilers from 28 to 49 d of age. Four different times of access (4, 8, 12 and 24 h) to the two alternate foods were compared. A whole wheat choice-feeding treatment and a complete single diet treatment were also compared. Whole wheat accounted for over 40% of the broilers' total food intakes when they were given the sequential feeding treatments of 8 h or greater. The whole wheat intakes of the birds given the 4 h sequential feeding and the choice-feeding were only 20 and 5% respectively. There was a non linear relationship between the weight gains of the broilers and the length of the sequential feeding period (P < 0.01). The growth rates of the broilers given sequential feeding were lowest (P < 0.05) in the 4-h feeding periods but highest (P < 0.05) in the 8-h periods. Weight gains decreased (P < 0.01) linearly as the sequential feeding periods were increased above 8 h. 3. A second trial, using 144 cage-reared broilers, examined the effect of different balancer compositions or different wheat varieties in 8-h sequential feeding. The broilers selected more whole wheat in their diet when they were given balancers with increased cereal contents. However, these broilers did not eat enough whole wheat to compensate for the reduced cereal content of the balancers and their overall diets had lower energy:protein ratios. The two different wheat samples did not result in any differences (P > 0.05) in the proportion of whole wheat selected by the broilers. 4. A third trial compared the diet selections, weight gains, food intakes and water excretions of 72 cage-reared broilers given whole wheat feeding regimens. The growth rates of the broilers given a loose mix of whole wheat and a pelleted balancer diet were similar (P > 0.05) to broilers given a complete single diet. The growth rates of these two groups were 7% greater (P < 0.05) than broilers given choice-feeding or 8-h sequential feeding.(ABSTRACT TRUNCATED AT 400 WORDS)